Example 2. whole genome comparison

Example files are contained in the Easyfig_example2_files folder (available to download from http://sourceforge.net/projects/easyfig/files/)

In this example we are creating a comparison figure of three _Escherichia coli _genomes, O157:H7 EDL933, O157H7 Sakai and K12 MG1655. We want to illustrate the phage regions, but as these are not labeled uniquely in the original GenBank files we have added them manually using "phage" as a feature label. For example in the K12 file we add 12 lines, one for each phage:

FEATURES             Location/Qualifiers  
     source          1..4639675  
                     /organism="Escherichia coli K12"  
                     /mol_type="genomic DNA"  
                     /strain="K-12"  
                     /sub_strain="MG1655"  
                     /db_xref="taxon:83333"  
     phage           262182..296489  
     phage           1195443..1210646  

etc…

The phage features have already been added to the files provided: EcSakai.withPhage.gbk, EcEDL933.withPhage.gbk, EcK12.withPhage.gbk.

A. Comparison of three _E. coli _genomes

1. Launch Easyfig. (If continuing from Example 1, quit Easyfig before starting)

2. LOAD ANNOTATION FILES as described in part 1A, in this order:

EcSakai.withPhage.gbk
EcEDL933.withPhage.gbk
EcK12.withPhage.gbk

3. LOAD BLAST COMPARISON FILES as described in part 1A, in this order:

Sakai_EDL933.blastn.out
EDL933_K12.blastn.out

4. SPECIFY OUTPUT FILE as described in part 1A

5. CREATE FIGURE as described in part 1A.

IMPORTANT: By default, Easyfig does not display features or BLAST matches smaller than 4 pixels. This feature can be turned off and on using the "Filter small blast hits/annotations" button found in the Blast option from the Image dropdown menu. If this filter is turned off with a whole genome sequence comparison the thousands of small, insignificant BLAST matches and features will swamp the image and greatly prolong rendering times. It is recommended that you turn this filter off and use the BLAST options and custom features (as described in Part B) to filter the small BLAST matches and features in whole genome comparisons.

B. Display custom features on a whole genome comparison

1. Access the annotation window by choosing annotation from the Image dropdown menu and uncheck the "gene" radio button under "Include following features"

2. Enter the word phage into the open custom feature box under "Include following features" and click the radio button on the right of the box

3. Click on Colour button ( … ) to the right of the custom feature box and change to your favourite colour

4. Use the pull-down menu to the far right of the custom feature box and choose "rect" for rectangle

5. Close the annotation window using the (close) button

6. Access the blast window by choosing Blast from the Image dropdown menu

7. Enter 10000 under "Min. length"

8. Unclick the "Filter small blast hits/annotations" button

9. CREATE FIGURE as described in part 1A.

C. Change the BLAST identity gradient and show a graph

1. In the Blast window from the Image dropdown menu, click the colour buttons ( … ) and choose colours for minimum and maximum colours for normal and inverted BLAST matches: normal-minimum: aqua
   normal-maximum: blue
   inverted-minimum: orange
   inverted-maximum: red
   (the exact colours you choose is not important, as long as there is a contrast between the normal and inverted BLAST matches)

2. Unclick the "Outline blast hits in black"

3. Access the Graph window by choosing graph from the Image dropdown menu

4. Choose "GC content" from the pull-down "Graph:" menu

5. CREATE FIGURE as described in part 1A.